Abstract
Outer membrane proteins (OMPs) in Gram-negative bacteria are essential for a number of cellular functions including nutrient transport and drug efflux. Escherichia coli BamA is an essential component of the OMP β-barrel assembly machinery and a potential novel antibacterial target that has been proposed to undergo large (~15 Å) conformational changes. Here, we explored methods to isolate anti-BamA monoclonal antibodies (mAbs) that might alter the function of this OMP and ultimately lead to bacterial growth inhibition. We first optimized traditional immunization approaches but failed to identify mAbs that altered cell growth after screening >3000 hybridomas. We then developed a “targeted boost-and-sort” strategy that combines bacterial cell immunizations, purified BamA protein boosts, and single hybridoma cell sorting using amphipol-reconstituted BamA antigen. This unique workflow improves the discovery efficiency of FACS + mAbs by >600-fold and enabled the identification of rare anti-BamA mAbs with bacterial growth inhibitory activity in the presence of a truncated lipopolysaccharide layer. These mAbs represent novel tools for dissecting the BamA-mediated mechanism of β-barrel folding and our workflow establishes a new template for the efficient discovery of novel mAbs against other highly dynamic membrane proteins.
Highlights
The emergence of multi-drug resistant bacteria is a global health crisis that demands the discovery of new antibiotics, which requires a better understanding of microbial physiology and vulnerabilities
After we established methods to produce high-quality recombinant E. coli BamA reconstituted into a non-detergent amphipol matrix, we set out to evaluate the impact of two commonly used adjuvants on the success of monoclonal antibodies (mAbs) discovery against this dynamic and essential outer membrane proteins (OMPs)
All polyclonal antibodies (pAbs) showed some FACS binding to the K-12 strain, suggesting that certain mAbs are able to bind BamA in the presence of the K-12 core-LPS (Fig. 1b)
Summary
The emergence of multi-drug resistant bacteria is a global health crisis that demands the discovery of new antibiotics, which requires a better understanding of microbial physiology and vulnerabilities. MAbs capable of functionally modulating an OMP into an activated or inhibited state have not been reported to date, and are likely to be rare Faced with these challenges, we endeavored to streamline anti-BamA mAb discovery by optimizing antigen formats (reconstitution matrices and native whole cells), adjuvants, immunization strategy, BamA-specific cell sorting, and high-throughput hybridoma purification. While our extensive traditional mAb discovery efforts failed to identify any functional anti-BamA mAbs, our optimized workflow improved the efficiency of FACS+ anti-BamA mAb generation by >600-fold and enabled the discovery of rare anti-BamA mAbs with direct growth inhibitory activity in the presence of a truncated LPS layer These growth inhibitory anti-BamA mAbs will enable deeper mechanistic studies of BamA function and further interrogation of BamA as a novel therapeutic target
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